Identification of Proteins Affecting Assembly, Regulation, and Function of Thylakoid Protein Complexes

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This research project will utilize the genomic sequence of the cyanobacterium Synechocystis sp. PCC 6803 (accessible through http://www.kazusa.or.jp/cyano/cyano.html) to functionally identify "new" proteins involved in the regulation, assembly and function of protein complexes that catalyze electron transfer in thylakoid membranes of this cyanobacterium. This project involves positive selection for spontaneous mutants (1) in which photosystem II activity is decreased or abolished, or (2) in which regulation of expression of particular genes has been modified. Positive selection will occur by screening for high-light tolerance of the Synechocystis sp. PCC 6803 strain that lacks photosystem I; this strain is sensitive to high light but can be propagated at high light intensity if photosystem II is impaired or if rates of electron transport processes in the thylakoid membrane or the regulation by particular redox components have been altered.The availability of the genomic DNA sequence of Synechocystis sp. PCC 6803 provides a powerful approach to a rapid identification of the location of spontaneous mutations that lead to high-light tolerance of the photosystem I-less strain. DNA from these positively selected mutants will be fragmented by restriction enzymes; each digest (treated with one enzyme) will be size-separated on a gel and gel slices (each reflecting a certain size range of DNA) will be tested for functional complementation of the original (high-light sensitive) strain from which the spontaneous mutant was derived. A complete restriction map of the Synechocystis sp. PCC 6803 genome has been constructed for 16 enzymes that cut this cyanobacterial genome 300-1000 times each. For each of the 16 restriction enzymes, the resulting fragments, identified through their genomic position, have been sorted by size and compiled into a table. Based on the size range of the fragments that can functionally complement for each restriction enzyme, an appropriate size range of restriction fragments can be selected from each table. These restriction fragments (each identified through their position on the genome) are pooled, and are then sorted according to the genomic nucleotide number at the end of each fragment. Upon inspection of this sorted list, clusters of nucleotide numbers can be identified. Generally only a single cluster can be found in which there is one representative from each of the selected size regions for each of the enzymes. By identifying the region that is common between all sequences in this cluster, the domain carrying the mutation of interest (generally not longer than 2 kbp) can be determined. The domain of interest will be amplified by PCR, the mutation will be determined, and mutations in unknown genes can thus be simply mapped and identified, even without cloning.Some of the complementing secondary mutations may be in known genes, but others will be in open reading frames for which no function is known yet. These open reading frames will be of particular interest as the results obtained will help to assign a role to these known sequences with unknown function. This method already has led to the identification of an open reading frame that is similar to ycf39 from chloroplasts and that affects photosystem II function in mutants. The next part of the project will be the preliminary analysis of the role of open reading frames that were identified. The types of analysis will depend on the open reading frame and on the phenotype of the mutant, but may involve the following: (1) sequence homology searches, (2) functional analysis of electron transport, (3) gene expression studies, and (4) protein synthesis and degradation measurements.This work will lead to the identification of currently unknown genes whose products may be involved in photosystem II assembly, in electron transfer out of the plastoquinone pool in thylakoid membranes, or in control of regulatory processes in the cell. This is an open field, but rapid progress will be made now the genomic sequencing of Synechocystis sp. PCC 6803 has been completed, enabling effective and comprehensive means of analysis such as the one described here. This area of research not only is an important complement to large-scale sequencing efforts, in that it provides information regarding the function of open reading frames, but also is expected to provide novel insights into the regulation mechanisms and assembly processes required for electron transport in thylakoid membranes.